Intra-acinar cell nuclear factor-κB (NF-κB) and trypsinogen activation are early events in secretagogue-induced acute pancreatitis. We have studied the relationship between NF-κB and trypsinogen activation in rat pancreas. CCK analogue caerulein induces early (within 15 min) parallel activation of both NF-κB and trypsinogen in pancreas in vivo as well as in pancreatic acini in vitro. However, NF-κB activation can be induced without trypsinogen activation by lipopolysaccharide in pancreas in vivo and by phorbol ester in pancreatic acini in vitro. Stimulation of acini with caerulein after 6 h of culture results in NF-κB but not trypsinogen activation. Protease inhibitors (AEBSF, TLCK, and E64d) inhibit both intracellular trypsin activity and NF-κB activation in caerulein stimulated acini. A chymotrypsin inhibitor (TPCK) inhibits NF-κB activation but not trypsin activity. The proteasome inhibitor MG-132 prevents caerulein-induced NF-κB activation but does not prevent trypsinogen activation. These findings indicate that although caerulein-induced NF-κB and trypsinogen activation are temporally closely related, they are independent events in pancreatic acinar cells. NF-κB activation per se is not required for the development of early acinar cell injury by supramaximal secretagogue stimulation.